Pseudomonas aeruginosa (PA) is the fourth most frequent cause of nosocomial infections and the leading pathogen associated with hospital acquired pneumonia and a major cause of acute respiratory failure. A common theme of most acute PA infections is the requirement for pre-existing epithelial cell injury. A number of in vitro and ex vivo studies support the hypothesis that injured epithelium is a preferred target for PA infection. Recently, we have discovered that PA can inhibit repair of injured lung epithelium in vitro through a mechanism that is dependent upon the bacterially secreted proteins ExoS and ExoT, thus perpetuating conditions that favor further colonization and host cell damage by PA. ExoS and ExoT are closely related proteins consisting of two domains: an N-terminal GAP domain with activity towards Rho family GTPases and a C-terminal ADPRT domain whose targets are unknown. Both domains are required for full activity. Our long term goals are to understand how PA interacts with eukaryotic cells. Our short term goals are to dissect the mechanism by which PA, particularly the secreted proteins ExoS and ExoT, antagonize repair of wounded lung epithelium. We will also test the roles of matrix metalloproteinases (MMPs) in vivo and in vitro in PA-mediated infections. These studies may lead to the development of new therapeutic, diagnostic, or preventative therapies. Furthermore, the use of PA as a "probe" of epithelial cell development and wound repair should also yield novel insights into this fundamentally important biological process. In aim 1, we will test the hypothesis that both the GAP and ADPRT domains of ExoS and ExoT are critical to the ability of these toxins to inhibit wound repair. Using a wide variety of techniques in conjunction with mutants in each or both domains, we will determine the function of each domain and the step at which it acts in preventing or delaying wound healing in several tissue culture models of injured epithelium. In aim 2, we will test the hypothesis that MMPs play an important role in alveolar lung epithelial repair in the physiological context of acute PA pneumonia. We will compare mortality, colonization, spread to distant organs, and lung histology of PA-infected wild type mice, mice pretreated with MMP inhibitors, and MMP-null mice. We will also measure in vitro wound healing before and after exposure to PA in alveolar lung epithelial type II cells derived from MMP-null and wild type mice. Better understanding of these complex behaviors may lead to new approaches to improve healing of lung injury from pneumonia, surgical incisions, and burn injuries as well as prevention of secondary infections from PA and other gram negative bacteria.